Osmolality of the mammalian kidney medulla is very high. The high osmolality provides driving force for water reabsorption and urinary concentration, a key function of the kidney for maintaining proper body fluid volume and blood pressure. Salt and urea are major solutes in the renal medullary interstitium. Unfortunately, high salt (hypertonicity) causes DNA damage and high urea causes cell death. For the successful function of the kidney, the renal medullary cells have to overcome the deleterious effects of hyperosmolality. The cells adapt to the hypertonicity by accumulating compatible osmolytes. We have shown that TonEBP play a central role in the compatible osmolyte accumulation via stimulating genes whose products either actively transport or synthesize compatible osmolytes. In addition, TonEBP stimulates the vasopressin regulated urea transporters that play a critical role in the counter current recycling of urea in the renal medulla. Our data demonstrate that TonEBP also stimulates a heat shock protein 70 that functions to protect the cells from the deadly stress of high urea. Thus, TonEBP controls a network of genes that is essential for the function and protection of the renal medulla. We have shown that nuclear localization is the major site of TnEBP regulation under physiological conditions of water diuresis and antidiuresis. We hypothesize that there are signaling pathways that control nucleocytoplasmic trafficking of TonEBP in response to changes in ambient tonicity. Aim 1 is to define domains of TonEBP responsible for tonicity-responsive changes in nuclear localization. Cultured cells will be used in combination with expression of mutated TonEBPs. We expect to define amino acids critical for nuclear import, nuclear export, and modulation of the two opposing activities. These domains will provide key reagents to uncover the tonicity signaling pathways. Phosphorylation of TonEBP temporally correlates with both nuclear important and export. Aim 2 is to define amino acid residues whose phosphorylation affects nuclear localization of TonEBP. Aim I is to uncover the role of protein-protein interaction in nuclear localization. We will investigate the role of TnEBP dimer formation by studying defective mutants. Other components of the TonEBP complex will be identified and their role will also be investigated. These studies are likely to uncover a new paradigm of signal transduction unique to the kidney medulla.